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The Power of Microarray
Technology
Ruth G. Alscher
Gene Expression:
Control Points
Responses to Environmental
Signals
Glycolysis, Citric Acid Cycle, and
Related Metabolic Processes
Free Radicals Attack Cells:
Survival Mechanisms?
ROS Arise as a Result of
Exposure to:
•
•
•
•
•
•
•
Ozone
Sulfur dioxide
High light
Paraquat
Extremes of temperature
Salinity
Drought
Free radicals arise throughout the
.
cell when stress is imposed
Wounding ,
Chilling
Pathogens
Ozone
Cell Wall
Pathogens
Wounding ,
Chilling
Ozone
Cell Wall
Mitochondrion
Post-transcriptional
Effects
Drought ,
Salinity
(ROS
subcellul ar
si tes
unclear)
Mitochondrion
Pos t-tra nscriptiona l
Effe cts
Drought ,
Salinity
Cytosol
Cytosol
Antioxidant
genes
Antioxidant
genes
Nucleus
Nucleus
Gene
(ROS
su bce llul ar
si tes
un cle ar)
Ex pression
Gene
Expression
Chloroplast
Chloroplast
Pos t-tra nscriptiona l
Effe cts
Post-transcriptional
Paraquat ,
Effects
High Light + Chilling
Sulfur Dioxide
Paraquat ,
High Light + Chilling
Sulfur Dioxide
,
,
Effects of Drought Stress on Gene
Expression in Loblolly Pine Trees
Virginia Tech: determining which genes are
essential for resistance to stress
Plant Biologists: Drs. Alscher and Chevone.,
Cecilia Vasquez
CS: Drs. Heath and Ramakrishnan, Margaret
Ellis, Logan Hanks
Statistics: Dr Key, Xiao Yang.
NC State (Forest Biotechnology): discovering new
genes in loblolly pine.
Investigating gene expression patterns in
stressed loblolly pine
Selected cDNAs are spotted on to a glass
surface (can be up to 20,000 different
sequences spotted on to one slide).
cDNAs derived from mRNA populations
obtained from treated or control tree are
hybridized to the cDNAs on the slide.
The Expression of Multiple Genes Can be
Visualized Simultaneously Using Microarrays
Relative Abundance
Detection
Detection
Treatment
1
1
1
Control
1
2
2
3
3
3
3
2
2
Mix
Spots: 1
2
(Sequences affixed to slide)
3
Hybridization
1
2
3
The Expression of Multiple Genes Can be
Visualized Simultaneously Using Microarrays
Iterative strategy for detection of genetic
interactions using microarrays
Detection of gene
expression effects
on microarrays
1
4
Genetic
Regulatory
Networks
Test mutant
phenotypes
3
Identify mutants
Characterize
2
gene function
Design of Microarrays
• Clones on the drought-stress microarrays were replicated and
randomly placed
• Experiment involved 384 archived pine ESTs
• Organized into 4 microtitre source plates after PCR
• Pipetted into 8 sets of 4 microtitre plates each
• Each set a different random arrangement of 384 ESTs
• Printed type A microarrays from first 4 sets
• Printed type B microarrays from second 4 sets
• Each array has 4 randomly placed replicates of each EST
• Each control versus stress comparison was done on 4 arrays — A
and B; flip dyes; A and B
• Total of 16 replicates of each EST in each comparison
Who’s Who
Computer Science
Plant Biology
Ruth Alscher
Plant Stress
Virginia Tech
Boris Chevone
Plant Stress
Dawei Chen
Molecular Biology
Bioinformatics
Ron Sederoff,
North
Ross Whetten
Carolina
Len van Zyl
State Univ. Y-H.Sun
Lenwood Heath (CS)
Algorithms
Virginia Tech
Naren Ramakrishnan (CS)
Data Mining
Problem Solving Environments
Craig Struble,
Vincent Jouenne (CS)
Image Analysis
Forest Biotechnology
Ina Hoeschele (DS)
Statistical Genetics
Keying Ye (STAT)
Bayesian Statistics
Statistics
Virginia Tech
Expresso
People
Ron Sederoff
Lenny Heath
Craig Struble
Ruth Alscher
Ross Whetten
Keying Ye
Boris Chevone
Y-H .Sun
Len van Zyl
Dawei Chen
Vincent Jouenne
Naren
Ramakrishnan
Hypotheses
• There is a group of genes whose expression
confers resistance to drought stress.
• Expression of this group of genes is lower under
severe than under mild stress.
• Individual members of gene families show
distinct responses to drought stress.
Selection of cDNAs for Arrays
• 384 ESTs (xylem, shoot tip cDNAs of loblolly)
were chosen on the basis of function and
grouped into categories.
• Major emphasis was on processes known to be
stress responsive.
• In cases where more than one EST had similar
BLAST hits, all ESTs were used.
Expresso: A Problem Solving
Environment for Microarray
Experiment Design and Analysis
• Integration of design and procedures
• Integration of image analysis tools and
statistical analysis
• Connections to web databases and sequence
alignment tools
• The software Aleph was used for inductive logic
programming (ILP).
Categories within Protective
and Protected Processes
Gene
Expression
Signal
Transduction
Protease-associated
ROS and Stress
Environmental
Change
Protective
Processes
Nucleus
Cell Wall Related
Trafficking
Phenylpropanoid
Pathway
Development
Protected
Processes
Secretion
Cells
Cytoskeleton
Tissues
Plant Growth Regulation
Chloroplast Associated
Metabolism
Carbon Metabolism
Respiration and Nucleic Acids
Mitochondrion
A Note about Categories
Categories are not mutually exclusive; gene(s)
may be assigned to more then one category. For
example, heat shock proteins have been
grouped under these different categories and
subcategories
– Abiotic stress – heat
– Gene expression – post-translational
processing – chaperones
– Abiotic stress - chaperones
Abiotic
Biotic
Stress
Protective
Processes
Cell Wall Related
“Isoflavone
Reductases”
Antioxidant
Processes
Phenylpropanoid
Pathway
Drought
Dehydrins, Aquaporins
Heat
Non-Plant
Heat shock proteins
(Chaperones)
Xenobiotics
GSTs
Chaperones
NADPH/Ascorbate/
Glutathione
Scavenging Pathway
Sucrose Metabolism
Cellulose
Categories
within
“Protective
Processes”
Arabionogalactan proteins
Cytosolic
ascorbate
peroxidase
superoxide
dismutase-Fe
superoxide
dismutase-Cu-Zn
glutathione
reductase
Extensins and proline rich proteins
Hemicellulose
Pectins
Xylose
Other Cell Wall Proteins
Lignin Biosynthesis
isoflavone reductases
phenylalanine ammonia-lyases
S-adenosylmethionine decarboxylases
glycine hydromethyltransferases
4-coumarate-CoA
ligases
CCoAOMTs
cinnamyl-alcohol
dehydrogenase
Quality Control
• Positive: LP-3, a loblolly gene known to
respond positively to drought stress in loblloly
pine, was included.
• LP-3 was positive in the moist versus mild
comparison, and unchanged in the moist versus
severe comparison.
• Negative: Four clones of human genes used as
negative controls in the Arabidopsis Functional
Genomics project were included. The clones did
not respond.
Spot and Clone Analysis
• Image Analysis: gridding, spot identification,
intensity and background calculation,
normalization
• Statistics:
• Fold or ratio estimation
• Combining replicates
• Higher-level Analysis:
• Clustering methods
• Inductive logic programming (ILP)
Current Status of Expresso
• Completely automated and integrated
– Statistical analysis
– Data mining
– Experiment capture in MEL
• Current Work: Integrating
– Image processing
– Querying by semi-structured views
– Expresso-assisted experiment composition
Future Directions
Next Generation Stress Chips
1. Time course, short and long term, to capture
gene expression events underlying
“emergency” and adaptive events following
drought stress imposition.
(Use all available ESTs for candidate stress
resistance genes.)
2. Generate cDNA library from stressed seedlings.
3. Initiate modeling of kinetics of drought stress
responses.
Microarray Data Analysis
How to use microarrays to learn more about the
influence of drought stress on gene expression?
Where the biologists need the computer scientists.
A. Confounding factors in the raw data
1. Limitations in accuracy (technique)
2. Biological variation (individuals)
B. How to apply corrections for these
confounding factors to maximize the
predictive power of the data.
C. Modeling regulatory networks.